Amino acid sequence and structure of Crotoxin B (PLA₂) from Crotalus durissus terrificus snake venom. (A) Amino acid sequence of Crotoxin B. The regions that originated the peptides 1, 2, and 3 are shown within the blue, orange, and red boxes, respectively. The residues from the catalytic site are highlighted in yellow, while the residues from the Ca²⁺ binding site are highlighted in green. The residues highlighted in purple are important for Crotoxin B toxicity/lethality, myotoxicity, edema induction, antibacterial, liposome-disrupting, enzymatic, and anticoagulant activities. (B) Structure of Crotoxin B (PDB: 2QOG) with the regions that originated peptides 1 and 2 highlighted with carbon atoms in green and peptide 3 highlighted with carbon atoms in purple.

Evaluation of Crotoxin B-derived peptides on cellular viability against TNBC cells and mammary benign cells. MDA-MB-231 (blue line) and MCF10A cells (black line) were treated with peptides 1 (a,d), 2 (b,e), and 3 (c,f) for 24 and 48 h at the concentration range of 0.2–20 µg/mL. Cellular viability assay was evaluated using the MTT method. Data are shown as mean ± SEM of at least three independent assays. Significant differences between control (0 µg/mL) and treated cells are designated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, according to two-way ANOVA and a Bonferroni post hoc test.

Activity of 3-NAntC (peptide 3) in breast cancer cell lines compared to benign cells. Tumor (MDA-MB-231 and MCF-7) and benign (HDFa, HMEC, and MCF10A) cell lines were treated with 3-NAntC for 24, 48, and 72 h at the concentration range of 0.2–1.0 µg/mL. Cellular viability assay was conducted using the MTT method. Data are shown as mean ± SEM of at least three independent assays in triplicate. (a–c) Benign cell lines treated with the same concentrations of 3-NAntC were compared to the control (0 µg/mL) using two-way ANOVA and a Bonferroni post hoc test. (d–f) MDA-MB-231 and MCF-7 tumor cell lines were treated with various concentrations of 3-NAntC and compared to the HMEC benign cell line using two-way ANOVA and a Bonferroni post hoc test, # p < 0.05, and #### p < 0.0001.

Comparison of cell viability between 3-NAntC and chemotherapy (doxorubicin and cisplatin). Cellular viability assays were performed on MDA-MB-231 (a–c) and HDFa (d–f) cell lines using the MTT method. Cells were treated with 3-NAntC, cisplatin, or doxorubicin for 24, 48, and 72 h at concentrations ranging from 0.2 to 2.0 µg/mL. Data are presented as mean ± SEM of at least three independent assays in triplicate. Significant differences between the control (0 µg/mL) and treated cells are designated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, according to two-way ANOVA and a Bonferroni post hoc test.

Antiproliferative effects of 3-NAntC in TNBC and normal cells. (a,b) Cellular proliferation assay using the BrdU incorporation method. MDA-MB-231 and HDFa cells were treated with 3-NAntC for 24, 48, and 72 h at a concentration range of 0.2–1.0 µg/mL. Data are shown as a mean ± SEM of at least three independent assays in triplicate. * p < 0.05, ** p < 0.01, and **** p < 0.0001 show significance compared to the control (0 µg/mL), and ## p < 0.01, ### p < 0.001, and #### p < 0.0001 show significance compared to the same concentration in HDFa cell lines according to the two-way ANOVA and a Bonferroni post hoc test. (c) Cell cycle progression assay. Cells were treated with 3-NAntC for 48 h at the concentration range of 0.2–0.8 µg/mL. A flow cytometry assay was conducted with propidium iodide labeling. Data are shown as a mean ± SEM of at least three independent assays. Significant differences between the control (0 µg/mL) and treated cells are designated as * p < 0.05, and ** p < 0.01, according to the two-way ANOVA and Dunnett post hoc test.

Effect of 3-NAntC in apoptosis/necrosis profile of the MDA-MB-231 cell line. (a–c) A flow cytometry with Annexin V and Propidium iodide labeling was performed. Cells were treated with the peptide 3-NAntC (0–0.8 µg/mL) or with doxorubicin (0.2 µg/mL) for 24, 48, and 72 h. 3-NAntC induced cell death by apoptosis with a very low amount of necrosis. Data are shown as a mean ± SEM of at least three independent assays. (d) LDH release assay. Cells were treated with the 3-NAntC peptide (0–1.0 µg/mL) for 24, 48, and 72 h and the LDH release was quantified. There is no significant difference in this biomarker, suggesting no involvement of either necrosis or pyroptosis as a primary cell death mechanism. Data are shown as mean ± SEM of at least three independent assays in triplicate. Significant differences between control (0 µg/mL) and treated cells are designated as * p < 0.05, *** p < 0.001, and **** p < 0.0001, according to the two-way ANOVA and Dunnett post hoc test.